Means for antibody characterization

ABSTRACT

The present disclosure provides means such as uses, nanoparticles, solutions, methods, kits and systems for screening target proteins for self-association properties (viscosity and opalescence) in ultra-dilute solutions. They provide means for screening a large number of target proteins at orders of magnitude lower concentrations than end-use formulations.

FIELD

The present disclosure provides means such as uses, nanoparticles,solutions, methods, kits and systems useful for detectingself-association characteristics of proteins (e.g., therapeuticantibodies) using ultra-dilute solution measurements.

BACKGROUND

Monoclonal antibodies (mAbs) are among the most successfulpharmaceutical modalities, used to treat a wide array of diseases.Despite their success, mAbs are prone to developability issues that posemajor manufacturing, stability, and delivery challenges. In particular,the development of mAbs intended for subcutaneous administration can behindered by the need for high concentration formulations, in which poorsolution behavior in the form of high viscosity, opalescence, phaseseparation, and aggregation are often limiting. While intravenousadministration of mAbs in hospital settings is routine, subcutaneousadministration, being minimally invasive, ensures greater patientcompliance and is readily adaptable in settings with inadequate medicalinfrastructures. Rapid selection of developable therapeutic mAbcandidates is always desired, but this urgency becomes even more acutewhen combating infectious disease pandemics. When developability issuesdo arise, resource intensive mitigation strategies must be employedwhich invariably cause delays and do not guarantee success. As such, itis far more efficient to select mAbs with favorable solution propertiesearly with significant emphasis being placed on identifying variantswith drug-like properties at the earliest stages of discovery.

SUMMARY

Disclosed herein is, in a first aspect, the use of a positively-chargedpolymer to stabilize a nanoparticle comprising a capture agent on thesurface.

Disclosed herein is, in a second aspect, a nanoparticle comprising onthe surface a capture agent and a positively-charged polymer.

Disclosed herein is, in a third aspect, a solution comprising aplurality of the nanoparticle of the second aspect.

Disclosed herein is, in a fourth aspect, a kit comprising a nanoparticleand a positively-charged polymer.

Disclosed herein is, in a fifth aspect, a method for determining thetendency of a target protein to self-associate, comprising the steps of

-   (i) capturing the target protein in a solution as defined in the    third aspect,-   (ii) determining the color of the solution,-   wherein a change in the color of the solution compared to a control    solution as defined in the third aspect without a target protein    indicates a tendency of the target protein to self-associate.

Disclosed herein are, in a sixth aspect, methods for screening a targetprotein in dilute concentrations for self-association properties,comprising: adsorbing a capture agent and a positively-charged polymeronto a surface (e.g., nanoparticle (e.g., a metal nanoparticle)) to forma capture agent conjugate; incubating the capture antibody conjugatewith the target protein in a solution (e.g., buffer) to form a targetprotein conjugate; measuring the absorbance of light of the targetprotein conjugate at multiple wavelengths ranging from 450 nm to about750 nm; and identifying a plasmon wavelength as the wavelength at whichthere is maximal absorbance by the target protein conjugate.

Disclosed herein are, in a seventh aspect, systems or kits comprisingone or more of each of a capture agent, a positively-charged polymer, acapture surface (e.g., a metal nanoparticle), a solution (e.g., buffer),a target protein, at least one calibration protein, and aspectrophotometer.

Other aspects and embodiments of the disclosure will be apparent inlight of the following detailed description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exemplary research and development processfor monoclonal antibodies. Antibody discovery campaigns begin with theidentification and verification of thousands of candidate antibodiesthat recognize the target biomolecule. The large pool is condensed tothe order of hundreds during the optimization phase, in which candidatesundergo affinity maturation and humanization. During these early stages,minute quantities (ng-μg range) of material are available, rendering theassessment of developability quality attributes such as viscosity,opalescence, and aggregation infeasible. Because of this, it is notuncommon for molecules with poor developability attributes to advanceinto clinical development, greatly increasing the amount of time andresources spent on generating a viable drug product. The assessment ofkey developability attributes during lead optimization using ultra lowconsumption, high throughput assays, such as the CS-SINS approachdescribed herein would facilitate the advancement of antibody candidateswith a reduced risk for complications during manufacture and formulationin later stages of development, accelerating timelines and reducingassociated resource expenditures.

FIGS. 2A-2D shows the evaluation of the stabilization of antibody-goldconjugates using polylysine. Immunogold conjugates from standard AC-SINSimplementations (FIG. 2A, left) were assessed for stability under commonformulation and process conditions (10 mM acetate and histidine buffers,pH 4.0-6.5). Through dynamic light scattering measurements, rapidaggregation of the conjugates was observed beginning at pH 5.0 andcontinuing through the relevant conditions up to pH 6.5 (FIG. 2B, left).A concomitant shift in the plasmon wavelength of the aggregatedconjugates was observed, measured by absorbance spectroscopy (FIG. 2C,left). The zeta potential was measured across the pH range and aconsistent decline in potential was observed as pH increased from 4.0,crossing zero near pH 5.75, and turning negative thereafter (FIG. 2D,left). To achieve immunogold conjugate stability in the formulationspace, a small amount (3% by mass) of polylysine, a highly chargedbiopolymer, was incorporated (FIG. 2A, center). Particle stabilizationwas observed up to pH 6.0 using both DLS measurements of particle size(FIG. 2B, middle) and optical measurements of plasmon wavelength (FIG.2C, middle). The zeta potential of the conjugates remained positiveacross the assayed pH range (FIG. 2D, middle). However, the absolutevalue of the potential near pH 6.0 was near zero, which is associatedwith aggregation in analogous experiments with non-stabilizedconjugates. Stabilization increased as polylysine was increasedthroughout the range of 3%-15%, as demonstrated by a highly stabilized15% polylysine condition (FIG. 2A, right). These conjugates were stableacross the relevant pH range, as indicated by DLS size measurements(FIG. 2B, right) and plasmon wavelength (FIG. 2C, right). The zetapotential of the highly stabilized particles was positive across themeasured range (FIG. 2D, right). FIG. 2E is an exemplary graph of theabsorbance as a function of wavelength used to determine the plasmonwavelength of the nanoparticles.

FIGS. 3A-3H show the application of charge-stabilized immunogoldconjugates for the assessment of weak protein-protein interactions. Adiverse panel of 56 monoclonal antibodies was employed to assess theutility of charge-stabilized immunogold conjugates in identifyingantibody self-interactions. The panel represented a faithfulrecapitulation of the clinical antibody landscape, as evidenced bysimilarities in the biophysical properties to a dataset of 500 moleculesextracted from the Therapeutic Antibody Database (TABS) (FIGS. 3A-3D).The robustness of the calibration approach (described in methods) wasdemonstrated by comparing the results of experiments performed withvarying concentrations of polylysine (3% vs. 10%). An excellentcorrelation was observed between the two conditions, demonstrating thatthe assay is resistant to influence from small changes in polylysineconcentration that may occur due to variability in reagent preparation(FIG. 3E). CS-SINS scores were compared to direct measurements ofcritical developability properties (See Kingsbury, J. S. et al. Sci Adv6, eabb0372, doi:10.1126/sciadv.abb0372 (2020), incorporated herein byreference in its entirety), which empirically established that mAbs withviscosity >30 cP or opalescence >12 NTU portend issues in manufactureand formulation during clinical development. Applying these benchmarks,it was shown that using a CS-SINS threshold of 0.35 led to theidentification of well behaved (viscosity <30 cP and opalescence <12NTU), viscous (>30 cP) or opalescent (>12 NTU), or problematic solutionbehavior in >85% of the mAb panel (FIGS. 3F-3H).

FIG. 4 shows the methodology of the CS-SINS calibration process. CS-SINSmeasurements are calibrated and evaluated using two tests. Only CS-SINSmeasurements that pass both tests should be accepted.

FIGS. 5A-5C show an exemplary calibration of CS-SINS measurements.Examples of experiments that passed (FIG. 5A) and failed (FIGS. 5B-5C)the calibration process due to slow mixing of the goat anti-human Fcantibody with the gold particles (FIG. 5B) or the addition of 50% of thetarget mAb concentration (FIG. 5C). To pass Test #1, the antibody-goldconjugates display plasmon wavelengths less than 534 nm (humanpolyclonal antibody) and 533 nm (NIST mAb). To past Test #2, theconjugates display linear fit parameters for the calibration panelversus the reference panel of mAbs that are within 10% of the idealvalues (1 for slope, 0 for intercept and 1 for R²). The CS-SINS scoresare calculated as described herein.

FIGS. 6A and 6B demonstrate that calibrated CS-SINS results are stronglycorrelated for conjugates prepared using different polylysineconcentrations. A robust calibration protocol was developed to normalizeresults from experiments with varying amounts of polylysine. Plasmonshift measurements for a panel of mAbs using goat anti-human Fc antibodyconjugates stabilized with polylysine at polylysine/IgG mass fractionsof 0.03 (97% IgG) and (90% IgG) (FIG. 6A). FIG. 6B is a graph of thecorrelation between the CS-SINS scores for the panel of mAbs measured atthe two polylysine/IgG mass fractions for the plasmon measurementsreported in FIG. 6A using the calibration detailed in FIGS. 4 and 5 .

FIGS. 7A-7C show that CS-SINS measurements are strongly correlated withdiffusion interaction parameters in an IgG subclass-specific behavior.CS-SINS scores (0.01 mg/mL) are well correlated with kD measurements(1-10 mg/mL) (FIG. 7A, top). CS-SINS scores are even better correlatedkD measurements for the subset of IgG1/IgG2 anti-bodies (FIG. 7A,middle) and less correlated for the subset of IgG4 antibodies (FIG. 7A,bottom). The solution behavior in the context of subclass reveals thatthe proposed 0.35 threshold value for CS-SINS measurements effectivelyidentifies all of the poorly behaved molecules in the IgG1/IgG2 subclassgrouping (FIG. 7B). The solution behavior for IgG4s is more difficult topredict as the majority are prone to high opalescence but do not alwaysrespond in the CS-SINS assay (FIG. 7C).

FIGS. 8A-8C are graphs of the evaluation of the use ofpolylysine-stabilized gold conjugates for measuring mAb self-associationin histidine formulations (pH 6). Gold particles were first coated withgoat anti-human Fc IgG and polylysine (≥70 kDa) (FIG. 8A), goatnon-specific IgG and polylysine (FIG. 8B), or only polylysine (FIG. 8C),and then the conjugates were incubated with a panel of mAbs withdifferent levels of self-association. To evaluate the dependence of theplasmon shifts on the amount of mAb adsorbed, the conjugates (after thefirst step conjugation step) were incubated with a constantconcentration of human antibody (0.01 mg/mL) that was composed of onlyhuman polyclonal antibody (0% mAb), only human mAb (100% mAb) orcombinations thereof. The reported plasmon shifts are relative to humanpolyclonal antibody. In FIGS. 8A and 8B, the mass ratio of polylysine toIgG was 0.03, and the concentrations of polylysine and IgG used duringconjugation were 0.012 and 0.388 mg/mL, respectively. In FIG. 8C, theconcentration of polylysine used during conjugation was 0.4 mg/mL.

FIGS. 9A and 9B are graphs showing the effect of polylysine size onCS-SINS measurements of plasmon shifts in histidine formulations (pH 6).Conjugates were prepared by co-adsorbing goat anti-human Fc antibodywith different size polylysine polymers, and then the conjugates wereused to measure plasmon shifts for a panel of human mAbs. FIG. 7A showsthe correlation between plasmon shifts measured using 30-70 kDapolylysine (0.03 polylysine/IgG mass ratio) and 70 kDa poly-lysine (0.03polylysine/IgG mass ratio). FIG. 7B shows the correlation betweenplasmon shifts measured using 15-30 kDa poly-lysine polymers (0.10polylysine/IgG mass ratio) and #70 kDa polylysine polymers (0.03polylysine/IgG mass ratio).

DETAILED DESCRIPTION

The present disclosure provides methods for predicting problematiccharacteristics (e.g., self-association) of protein formulations usingsmall protein amounts in ultra-dilute solutions.

Antibodies with low levels of self-association present a reduced risk ofunfavorable solution behavior during manufacturing, formulation, anddelivery at high concentrations. While dilute-solution interactionspresent an effective approach for predicting antibody solution behavior,it is challenging to employ relevant screening methods for large numbersof antibodies during early discovery (FIG. 1 ). Traditional techniquesrequire relatively concentrated protein solutions (>1 mg/mL) for makingsuch measurements. This single challenge related to antibodyconcentration has prevented large and systematic analyses of antibodyself-association during antibody lead optimization (FIG. 1 ). Assayingantibody interactions at much lower concentrations (1-10 μg/mL) in amanner predictive of high concentration solution behavior (e.g.,viscosity at 150 mg/mL) would enable the selection of well-behaved mAbsfrom a much larger pool of candidates in early discovery.

A previously used approach (affinity-capture nanoparticle spectroscopy,AC-SINS) involved adsorbing anti-human capture antibodies on goldnanoparticles (20 nm) and using the conjugates to capture human mAbs ofinterest. The capture of multiple mAbs in proximity amplified colloidalinteractions and led to sensitive detection of antibodyself-interactions via measurable changes in optical properties inherentto gold nanoparticles. However, a key limitation of AC-SINS was that itwas not compatible with process streams and formulation conditions inthe pH 4.5-7 range and low ionic strength, commonly employed duringantibody development. Gold nanoparticles coated with capture(anti-human) antibodies were unstable under such conditions and readilyaggregate.

Described herein is development of systems and methods referred to ascharge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS)that overcame the limitations of AC-SINS by stabilizing theantibody-gold conjugates using positively-charged polymers. Moreover,CS-SINS was capable of robustly evaluating mAb weak, self-interactionsat ultra-dilute concentrations (10 μg/mL) that predict problematic highviscosity and opalescence of antibodies at four orders of magnitudehigher concentrations (150 mg/ml).

Section headings as used in this section and the entire disclosureherein are merely for organizational purposes and are not intended to belimiting.

1. DEFINITIONS

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. For example,any nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those that are well known and commonly used in the art. Themeaning and scope of the terms should be clear; in the event, however ofany latent ambiguity, definitions provided herein take precedent overany dictionary or extrinsic definition. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

The term “antibody,” as used herein, refers to a protein that isendogenously used by the immune system to identify and neutralizeforeign objects, such as bacteria and viruses. Typically, an antibody isa protein that comprises at least one complementarity determining region(CDR). The CDRs form the “hypervariable region” of an antibody, which isresponsible for antigen binding (discussed further below). A wholeantibody typically consists of four polypeptides: two identical copiesof a heavy (H) chain polypeptide and two identical copies of a light (L)chain polypeptide. Each of the heavy chains contains one N-terminalvariable (V_(H)) region and three C-terminal constant (C_(H1), C_(H2),and C_(H3)) regions, and each light chain contains one N-terminalvariable (V_(L)) region and one C-terminal constant (C_(L)) region. Thelight chains of antibodies can be assigned to one of two distinct types,either kappa (κ) or lambda (λ), based upon the amino acid sequences oftheir constant domains. In a typical antibody, each light chain islinked to a heavy chain by disulfide bonds, and the two heavy chains arelinked to each other by disulfide bonds. The light chain variable regionis aligned with the variable region of the heavy chain, and the lightchain constant region is aligned with the first constant region of theheavy chain. The remaining constant regions of the heavy chains arealigned with each other. The variable regions of each pair of light andheavy chains form the antigen binding site of an antibody. The V_(H) andV_(L) regions have the same general structure, with each regioncomprising four framework (FW or FR) regions. The term “frameworkregion,” as used herein, refers to the relatively conserved amino acidsequences within the variable region which are located between the CDRs.There are four framework regions in each variable domain, which aredesignated FR1, FR2, FR3, and FR4. The framework regions form the βsheets that provide the structural framework of the variable region(see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., GarlandPublishing, New York, N.Y. (2001)). The framework regions are connectedby three CDRs. As discussed above, the three CDRs, known as CDR1, CDR2,and CDR3, form the “hypervariable region” of an antibody, which isresponsible for antigen binding. The CDRs form loops connecting, and insome cases comprising part of, the beta-sheet structure formed by theframework regions. While the constant regions of the light and heavychains are not directly involved in binding of the antibody to anantigen, the constant regions can influence the orientation of thevariable regions. The constant regions also exhibit various effectorfunctions, such as participation in antibody-dependentcomplement-mediated lysis or antibody-dependent cellular toxicity viainteractions with effector molecules and cells.

The terms “fragment of an antibody,” “antibody fragment,” and“antigen-binding fragment” of an antibody are used interchangeablyherein to refer to one or more fragments of an antibody that retain theability to specifically bind to an antigen (see, generally, Holliger etal., Nat. Biotech., 23(9): 1126-1129 (2005)). Any antigen-bindingfragment of the antibody described herein is within the scope of theinvention. The antibody fragment desirably comprises, for example, oneor more CDRs, the variable region (or portions thereof), the constantregion (or portions thereof), or combinations thereof. Examples ofantibody fragments include, but are not limited to, (i) a Fab fragment,which is a monovalent fragment consisting of the V_(L), V_(H), C_(L),and C_(H), domains, (ii) a F(ab′)₂ fragment, which is a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region, (iii) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (iv) a Fab′ fragment, whichresults from breaking the disulfide bridge of an F(ab′)₂ fragment usingmild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv),and (vi) a domain antibody (dAb), which is an antibody single variableregion domain (V_(H) or V_(L)) polypeptide that specifically bindsantigen.

A “spectrophotometer” is any variety of instrument that measure lightabsorption and/or transmission at a variety of light wavelengths. Aspectrophotometer is most commonly applied to ultraviolet (185-400 nm),visible (400-700 nm), and infrared (700-15000 nm) radiation, but somespectrophotometers can interrogate wide swaths of the electromagneticspectrum, including x-ray, ultraviolet, visible, infrared, and/ormicrowave wavelengths. Spectrophotometers have a light source, amonochromator, a wavelength selector, a sample holder, and a detector.Any spectrophotometer capable of processing solution-based samples maybe used, including those with cuvette sample holders, plater readers,and the like.

A “polypeptide”, “protein,” or “peptide” is a linked sequence of two ormore amino acids linked by peptide bonds. The polypeptide can benatural, synthetic, or a modification or combination of natural andsynthetic. Peptides and polypeptides include proteins such as bindingproteins, receptors, and antibodies. The proteins may be modified by theaddition of sugars, lipids or other moieties not included in the aminoacid chain. The terms “polypeptide”, “protein,” and “peptide” are usedinterchangeably herein.

Positively-charged polymers can be any of a variety of compounds havinga net positive charge. Positively-charged polymers useful in the presentinvention include positively charged peptides and proteins, bothnaturally occurring and synthetic, as well as polyamines, carbohydratesor synthetic polycationic polymers. The positively-charged polymers maybe linear or branched polymers, and may have interconnection betweenrepeating units in addition to the main chain linkages.Positively-charged polymer compositions may have any level ofpolydispersity from substantially monodisperse to substantiallypolydisperse. A substantially monodisperse composition comprises polymermolecules, substantially all of which have the same chain length. Asubstantially polydisperse composition comprises polymer molecules witha variety of chain lengths (and hence molecular weights).

The concentration of the positively-charged polymer may vary but isdirected to those concentrations which prevent self-aggregation of thecapture agent conjugate in the absence of a target protein. Aggregationof the capture agent conjugate can be measured under a variety ofsolution conditions (pH, conjugate concentration, temperature, ionicstrength) by a number of methods known in the art, including forexample, dynamic light scattering as demonstrated herein.

The concentration of the positively-charged polymer may be greater thanabout 0.1% by mass of positively-charged polymer and capture agent (i.e.0.1% positively-charged polymer and 99.9% capture agent), greater thanabout 1% by mass of positively-charged polymer and capture agent,greater than about 3% by mass of positively-charged polymer and captureagent, greater than about 5% by mass of positively-charged polymer andcapture agent, greater than about 10% by mass of positively-chargedpolymer and capture agent, greater than about 15% by mass ofpositively-charged polymer and capture agent, greater than about 20% bymass of positively-charged polymer and capture agent, greater than about30% by mass of positively-charged polymer and capture agent, greaterthan about 40% by mass of positively-charged polymer and capture agent.In some embodiments, the positively-charged polymer is added at aconcentration greater than 3% by mass of positively-charged polymer andcapture agent.

The concentration of the positively-charged polymer may be between about0.1% to about 50% by mass of positively-charged polymer and captureagent. The concentration of the positively-charged polymer may bebetween about 0.1% to about 50%, between about 1% to about 50%, betweenabout 3% to about 50%, between about 5% to about 50%, between about 10%to about 50%, between about 20% to about 50%, between about 30% to about50%, between about 0.1% to about 40%, between about 1% to about 40%,between about 3% to about 40%, between about 5% to about 50%, betweenabout 10% to about 50%, between about 20% to about 50%, between about30% to about 50%, between about 0.1% to about 30%, between about 1% toabout 30%, between about 3% to about 30%, between about 5% to about 30%,between about 10% to about 30%, between about 20% to about 30%, betweenabout to about 20%, between about 1% to about 20%, between about 3% toabout 20%, between about 5% to about 20%, between about 10% to about20%, between about 0.1% to about 15%, between about 1% to about 15%,between about 3% to about 15%, between about 5% to about 15%, betweenabout 10% to about 15%, between about 0.1% to about 10%, between about1% to about 10%, between about 3% to about 10%, between about 5% toabout 10%, between about 0.1% to about 5%, between about 1% to about 5%,between about 3% to about 5%, between about 0.1% to about 3%, betweenabout 1% to about 3%, or between about to about 1% by mass ofpositively-charged polymer and capture agent. In some embodiments, thepositively-charged polymer is added at a concentration greater than 3%by mass of positively-charged polymer and capture agent. In someembodiments, the positively-charged polymer is added at a concentrationbetween about 3% and about 15% by mass of positively-charged polymer andcapture agent.

Positively-charged polymers can have a wide range of molecular weights.In some embodiments, a positively-charged polymers can have a molecularweight greater than about kDa, greater than about 15 kDa, greater thanabout 20 kDa, greater than about 25 kDa, greater than about 30 kDa,greater than about 40 kDa, greater than about 50 kDa, or greater thanabout 60 kDa, greater than about 70 kDa, greater than about 80 kDa,greater than about kDa, greater than about 100 kDa, greater than about150 kDa, greater than about 200 kDa, or greater. In other embodiments,the positively-charged polymers can have a molecular weight between10-500 kDa, between 10-250 kDa, between 10-200 kDa, between 15 and 70kDa, between 30 and 70 kDa, or between 15 and 30 kDa. However, othersizes may be used which prevent self-aggregation of the capture agentconjugate. Molecular weights can be determined by those of ordinaryskill in the art by methods such as size-exclusion chromatography and/ormulti-angle laser light scattering techniques.

In certain embodiments of the invention the positively-charged polymermay fall under the class of synthetic polypeptides, also known aspolyamino acids. A synthetic polypeptide may be a homopolymer of one ofthe positively charged (i.e., basic) amino acids such as lysine,arginine, or histidine, or a heteropolymer of two or more positivelycharged amino acids. In some embodiments, the polycation may bepolylysine (e.g., poly-D-lysine, poly-L-lysine, and poly-DL-lysine),polyarginine, and polyhistidine, in particular polylysine. In addition,the polymer may comprise one or more positively charged non-standardamino acids such as ornithine, 5-hydroxy lysine, and the like. Or, thepolypeptide may be functionalized with other groups, such aspoly(y-benzyl-L-glutamate). Any of the combination amino acids can bepolymerized into linear, branched, or cross-linked chains. Suchpolycationic polypeptides may contain at least 50 amino acid residues,at least 100 amino acid residues, at least 200 amino acid residues, atleast 300 amino acid residues, at least 500 amino acid residues, atleast 750 amino acids, at least 1000 amino acids, at least 2000 aminoacids, at least 3000 amino acids, at least 4000 amino acids or more(e.g., from about 50 to about 500 amino acid residues, from about 50 toabout 1000 amino acid residues, or from about 100 to about 1000 aminoacid residues). Synthetic polypeptides can be produced by methods knownto those of ordinary skill in the art, for example, by chemicalsynthetic methods or recombinant methods. In select embodiments, thepositively-charged polymer is polylysine of greater than or equal toabout 70 kDa. In select embodiments, the positively-charged polymer ispolylysine added at a concentration of 3-15% weight to volume.

In some embodiments, the positively-charged polymers comprise syntheticpolycationic polymers, including but not limited to polyethylenimine(PEI), polyamidoamine (PAMAM), and the like.

The term “stabilizing” with regard to nanoparticles refers in particularto a prevention or at least reduction of aggregation of nanoparticles.“Aggregation” refers to formation of assemblages in a suspension andrepresents a mechanism leading to the functional destabilization ofcolloidal systems. During this process, particles dispersed in theliquid phase stick to each other, and spontaneously form irregularparticle assemblates, flocs, or agglomerates. This phenomenon is alsoreferred to as coagulation or flocculation and such a suspension is alsocalled unstable.

As used herein, the term “nanoparticle” refers to small particles havinga size (e.g. diameter) on the scale of 0.001 μm to 1 μm. Nanoparticlesmay be in the form of spheres, rods, chains, stars, flowers, reefs,whiskers, fibers, boxes, and the like. The size refers to the largestdistance from one point of the nanoparticle to another, e.g. for spheresit is the diameter.

Nanoparticles may comprise any material, including metals, semiconductormaterials, magnetic materials, and combinations of materials.Specifically envisaged with regard to the present disclosure are metalnanoparticles, wherever referred to herein. Metal nanoparticles, e.g.,gold or silver nanoparticles, are inherently suitable for surfaceplasmon resonance-based assays, since metal spheres have free electronson their surface that can interact with the electric field from incidentlight, resulting in a strong absorbance spectrum. In some embodiments,the nanoparticle comprises a gold nanoparticle, has a gold surface orconsists of gold. The size of the nanoparticle may affect the intensityand wavelength of maximum absorbance. In some embodiments, thenanoparticles have a diameter from 1 nm to 1000 nm, e.g., 1 nm to 200nm. In some embodiments, the nanoparticle is 5 nm to 50 nm in size(e.g., diameter), such as approximately 20 nm. In exemplary embodiments,the nanoparticle comprises or is a 20 nm gold nanoparticle. The overallquantity of the nanoparticles in the assay may affect the signal tonoise measurement. In some embodiments, 20 nm gold nanoparticles areused at a concentration of at least 7.0×10⁹ particles/mL. In someembodiments, 20 nm gold nanoparticles are used at a concentration of7.0×10⁹-1.5×10⁹ particles/mL (e.g., 1.0×10¹⁰-1.5×10⁹ particles/mL).

A capture agent is any agent that is capable of binding to a targetprotein (“capturing a target protein”, specifically in a solution asdefined in the third aspect). Thus, the nature of the capture agent willdepend on the type of target protein. For example, the capture agent maycomprise a binding partner of the target protein, either natural orsynthetic, including proteins, nucleic acids, carbohydrates, smallmolecules, or another binding moiety recognized specifically by thetarget protein. In some embodiments, the capture agent comprises anantibody, or a derivative or fragment thereof, capable of binding thetarget protein. The capture agent can be directly adsorbed to thenanoparticle, or alternatively, the nanoparticle may comprise a linkerthat tethers the capture agent to the nanoparticle. In some embodiments,the capture agent is a protein or a protein ligand. In specificembodiments, it is (or comprises) an antibody or a protein comprising anantigen-binding fragment of an antibody. The antibody can in particularbe an Fc-specific antibody (i.e. an antibody binding to the Fc portionof another antibody), such as an IgG-Fc-specific antibody, specificallyan IgG1-, IgG2- or IgG4-Fc-specific antibody, and more specifically anIgG1- or IgG4-Fc-specific antibody. In some embodiments, the antibody isan anti-human antibody. A target protein may be any protein in which onedesires to determine self-association or self-aggregation propertiesusing small quantities of protein in dilute solutions. In someembodiments, the target protein comprises a therapeutic protein.Therapeutic proteins comprise both purified and synthetic proteinsuseful for the treatment of diseases and disorders in a subject.Therapeutic proteins may include, but are not limited to, antibody-baseddrugs, Fc fusion proteins, anticoagulants, blood factors, bonemorphogenetic proteins, engineered protein scaffolds, enzymes, growthfactors, hormones, interferons, interleukins, and thrombolytics. In someembodiments, the target protein comprises an antibody or a fragment or aderivative thereof. In select embodiments, the target protein comprisesan antibody, specifically a monoclonal antibody, more specifically ahuman monoclonal antibody. In one embodiment, the antibody is an IgGantibody, for example an IgG1, IgG2 or IgG4 antibody. In a selectembodiment thereof, the antibody is an IgG1 or IgG2 antibody.

The target protein may be at any concentration. In the case oftherapeutic proteins, the concentration will likely be considerably moredilute than that of the therapeutic formulation. In some embodiments,the target protein is at a concentration of less than 1 mg/mL (e.g.,less than 500 μg/mL, less than 100 μg/mL, less than 50 μg/mL, or lessthan 10 μg/mL). The target protein may be at a concentration of 1-1000μg/mL, 1-100 μg/mL, 1-50 μg/mL, 1-20 μg/mL, or 1-10 pg/mL. In someembodiments, the target protein is at a concentration of 1-10 μg/mL. Insome embodiments, the target protein is in a solution (e.g., buffer)with a pH value of 3.5-7 (e.g., 4.5-7), specifically 4-6.5 or 4-6. Insome embodiments, the solution (e.g. buffer) has low ionic strength.

Ionic strength is a measure of the concentrations of ions in solution.The term “low ionic strength solution” refers to a solution like abuffer with a total buffer agent concentration of less than about 50 mM,whereas high ionic strength buffers are those with a concentrationgreater than about 100 mM. Buffer agents are well known in the art andcan be salts of a weak acid and a weak base. Examples are carbonates,bicarbonates, and hydrogen phosphates. In specific embodiments of thedisclosure, the buffer is a histidine and/or acetate buffer or a bufferhaving substantially the same ionic strength as a histidine and/oracetate buffer. A histidine and/or acetate buffer may have aconcentration of less than 50 mM histidine and/or 50 mM acetate, e.g.,less than 25 mM histidine and/or 25 mM acetate, specifically about 10 mMhistidine and/or 10 mM acetate. “Substantially the same” means ±50%,±40%, ±30%, ±20%, ±10%, or ±5%.

The term “conjugation” or “conjugating to” refers to the bond ofmolecules to nanoparticles by chemical, physical or biological means. Aconjugation of nanoparticles to a molecule is usually meant whenreferring to a nanoparticle comprising a molecule on its surface. Aspecific meaning of the term is “adsorption” or “adsorbing onto”, whichincludes physisorption (van der Waals forces), chemisorption (covalentbonding) and electrostatic attraction. In a select embodiment, it isphysisorption.

The term “self-association” refers to the interaction between the samekind of protein (e.g., antibodies) and a multimer formation. Suchmultimer formation may return to the monomer by an operation such asdilution. The “tendency” for self-association refers to the ease ofself-association, in particular by increasing the concentration of theprotein, e.g., above 50 mg/ml, 100 mg/ml or 150 mg/ml (referred to as“high” or “higher” concentration herein). An example of a parameterindicating self-association is the diffusion interaction parameter. Thediffusion interaction parameter is an index of self-associationcalculated using the concentration dependence of the diffusioncoefficient obtained by an experimental method. If the value is −12.4g/mL or more, repulsive force between molecules predominates. If it isless than that, it is reported to be an attractive (self-associative)interaction (Saito et al., Pharm. Res., 2013. Vol. 30 p1263). Thediffusion coefficient is an index of the ease of diffusion of moleculesin a solution and can be measured by a dynamic light scattering methodor the like. More generally, protein self-association may result in poorsolution behavior including high viscosity, opalescence, phaseseparation and/or aggregation.

The term “plasmon wavelength” refers to the wavelength of maximumabsorbance.

Preferred methods and materials are described below, although methodsand materials similar or equivalent to those described herein can beused in practice or testing of the present disclosure. All publications,patent applications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

2. USES FOR STABILIZING NANOPARTICLES

In a first aspect, the disclosure relates to the use of apositively-charged polymer to stabilize a nanoparticle comprising acapture agent on the surface.

The capture agent is usually conjugated to the nanoparticle. In specificembodiments, the nanoparticle is comprised in a solution. Accordingly,the use may be for stabilizing nanoparticles, in particular a pluralitythereof, in a solution (i.e. for stabilizing a nanoparticle suspension).The solution may be one as defined in the third aspect below.

The use usually comprises conjugating the positively-charged polymer tothe nanoparticle before, at the same time, or after conjugating thecapture agent to, the nanoparticle, in one specific embodiment at thesame time. Concentrations (by mass of positively-charged polymer andcapture agent) of the positively-charged polymer can be as describedabove. In some embodiments, the use comprises determining thestabilization of the nanoparticle by determining a plasmon wavelength ofthe nanoparticle in solution while capturing a human polyclonal antibody(e.g., human polyclonal antibody ChromPure Human IgG) of 534 nm or less,and/or a plasmon wavelength of the nanoparticle in solution whilecapturing NIST monoclonal antibody (reference material RM8671) mAb of533 nm or less.

3. NANOPARTICLES

In a second aspect, the disclosure relates to a nanoparticle comprisingon the surface a capture agent and a positively-charged polymer.

In some embodiments, the nanoparticle is a metal nanoparticle,specifically a gold nanoparticle.

In some embodiments, the capture agent and/or the positively-chargedpolymer are conjugated to the nanoparticle. The amount ofpositively-charged polymer (by mass of positively-charged polymer andcapture agent) can be as described above. In a specific embodiment, thenanoparticle is a stable nanoparticle, i.e. stabilized, for exampleaccording to the use of the first aspect. Specifically, the plasmonwavelength of the nanoparticle in solution while capturing a humanpolyclonal antibody (e.g., human polyclonal antibody ChromPure HumanIgG) is 534 nm or less, and/or the plasmon wavelength of thenanoparticle in solution while capturing NIST monoclonal antibody(reference material RM8671) mAb is 533 nm or less.

4. SOLUTIONS COMPRISING NANOPARTICLES

In a third aspect, the disclosure relates to a solution comprising aplurality of the nanoparticle of the second aspect. As such, thesolution may also be termed a nanoparticle suspension.

In some embodiments, the solution is a buffer, specifically of a lowionic strength. The pH of the solution/buffer may be 3.5-7 (e.g.,4.5-7), specifically 4-6.5 or 4-6. A plurality means at least 2, atleast 10, at least 100, or at least 1000.

It is to be understood that wherever this disclosure refers to a use ofa nanoparticle of the second aspect, it is intended to also refer to theuse of the solution of the third aspect instead.

5. KITS COMPRISING NANOPARTICLES

In a fourth aspect, the disclosure relates to a kit comprising ananoparticle (in particular a plurality thereof) and apositively-charged polymer.

The kit may further comprise a solution like a buffer, in particular alow ionic strength solution/buffer. The pH of the solution/buffer may be3.5-7 (e.g., 4.5-7), specifically 4-6.5 or 4-6. In further embodiments,the kit may also comprise (i) a capture agent, optionally conjugated tothe nanoparticle, and/or (ii) a panel of at least 2, e.g., at least 3,4, 5 or 6, calibration proteins. The kit may in addition compriseinstructions for use providing the plasmon wavelength of the calibrationproteins of the panel of calibration proteins. These plasmon wavelengthsare known or “historical” plasmon wavelengths of the calibrationproteins.

6. METHODS FOR DETERMINING SELF-ASSOCIATION OF A TARGET PROTEIN

In a fifth aspect, the disclosure relates to a method for determiningthe tendency of a target protein to self-associate, comprising the stepsof

-   (i) capturing the target protein in a solution as defined in the    third aspect,-   (ii) determining the color of the solution, wherein a change in the    color of the solution compared to a control solution as defined in    the third aspect without a target protein indicates a tendency of    the target protein to self-associate.

The control solution may be the solution before step (i) and before thetarget protein is added, or it may be a separate solution (e.g.,undergoing the same method with the exception of step (i)).

The concentration of the target protein may be as described above fortarget protein analysis. The step of capturing may comprise mixingand/or incubating (e.g., as described below with regard to the sixthaspect) the solution.

The change in color is usually towards a higher absorption wavelength.In some embodiments, determining the color comprises determining thelight absorption, wherein a change in the light absorption compared tothe control solution indicates a tendency of the target protein toself-associate. In specific embodiments, step (ii) comprises determiningthe plasmon wavelength, wherein a change in the plasmon wavelengthcompared to the control solution indicates a tendency of the targetprotein to self-associate. The determination may comprise measuring theabsorbance of light at multiple wavelengths ranging from 450 nm to about750 nm to determine the plasmon wavelength. The wavelength correspondingto maximal absorbance (plasmon wavelength) shifts to greater values asthe separation distance between nanoparticles is reduced. Thus, as thetarget protein self-associates, the absorbance spectrum of thenanoparticles changes and the plasmon wavelength increases. For example,when the nanoparticle is a gold nanoparticle, a color change towardsred, e.g., a red shift in plasmon wavelength indicates a tendency of thetarget protein to self-associate. Usually, the extent of the change ofcolor, light absorption or plasmon wavelength, respectively, indicatesthe strength of the tendency of the target protein to self-associate.However, in some embodiments, the change can be used as a binaryindication of self-association and no self-association, based on whetherthe plasmon wavelength changes due to capturing of the target protein.

The solution is usually a solution as defined in the third aspect. Thenanoparticle in solution (or the solution) has, in a select embodiment,a plasmon wavelength while capturing a human polyclonal antibody (e.g.,human polyclonal antibody Chrom Pure Human IgG) of 534 nm or less,and/or a plasmon wavelength while capturing NIST monoclonal antibody(reference material RM8671) mAb of 533 nm or less. To this end, themethod may comprise prior to step (i) a step of selecting a nanoparticle(or solution), wherein the plasmon wavelength of the nanoparticle insolution while capturing a human polyclonal antibody (e.g., humanpolyclonal antibody ChromPure Human IgG) is 534 nm or less, and/or theplasmon wavelength of the nanoparticle in solution while capturing NISTmonoclonal antibody (reference material RM8671) mAb is 534 nm or less,for the capturing in step (i).

In some embodiments, the method comprises a step of calibrating using apanel of at least 2, preferably at least 3, 4, 5 or 6, calibrationproteins (e.g., antibodies, specifically monoclonal antibodies) havingdifferent tendencies to self-associate. These tendencies are known. Thecalibrating may comprise the steps of:

-   (i) measuring the plasmon wavelength of each calibration protein of    the panel,-   (ii) calculating a historical CS-SINS score for each calibration    protein (cP) according to the following formula: CS-SINS    Score=(cP—parameter 1)/(parameter 2—parameter 1), wherein parameter    1 is the plasmon wavelength of the calibration protein with the    lowest tendency to self-associate of the panel and parameter 2 is    the plasmon wavelength of the calibration protein with the highest    tendency to self-associate of the panel,-   (iii) calculating a new CS-SINS score for each calibration protein    according to the following formula: CS-SINS Score=(cP—parameter    1)/(parameter 2—parameter 1), wherein parameter 1 is the plasmon    wavelength of the calibration protein with the lowest tendency to    self-associate of the panel and parameter 2 is the plasmon    wavelength of the calibration protein with the highest tendency to    self-associate of the panel, and fitting the parameters to maximize    the agreement of the linear fit between the historical and new    parameters by minimizing the following term:    ((1-slope)²+(intercept)²)),-   (iv) determining whether the calibration meets the following    criteria a)-c) for a linear fit between the new and the historical    data:    -   a) slope of linear fit is between 0.9 and 1.1,    -   b) intercept of linear fit id between −0.1 and 0.1,    -   c) R² of linear fit is above 0.9; and-   (v) if all criteria of step (iv) are met, calibrating the CS-SINS    score of the target protein using the fitted parameters identified    in step (iii). Step (ii) can be carried out before, after or at the    same time as step (i). In a specific embodiment, a target protein    CS-SINS score of less than or equal to about 0.35 indicates that the    target protein has a low tendency to self-associate (i.e. has    favorable self-association properties for formulations and    compositions comprising high concentrations of the target protein    (e.g., greater than or equal to 100 mg/mL target protein)).

7. METHODS FOR SCREENING A TARGET PROTEIN

The present disclosure provides methods for screening a target proteinin dilute concentrations for self-association properties (e.g.,opalescence and viscoelastic properties). Generally, the use of themethod of the fifth aspect is provided for screening target proteins fortheir tendency to self-associate. More specifically, in a sixth aspect,the disclosure relates to methods that may comprise adsorbing a captureagent and a positively-charged polymer onto a nanoparticle (e.g., ametal nanoparticle) to form a capture agent conjugate; incubating thecapture antibody conjugate with the target protein in a solution (e.g.,buffer) to form a target protein conjugate; measuring the absorbance oflight of the target protein conjugate at multiple wavelengths rangingfrom 450 nm to about 750 nm; and identifying a plasmon wavelength as thewavelength at which there is maximal absorbance by the target proteinconjugate.

The positively-charged polymer is co-adsorbed onto a surface (e.g.,nanoparticle) with the capture agent

The methods may comprise incubating the capture antibody conjugate withthe target protein in a solution (e.g., buffer) to form a target proteinconjugate. The incubation may be any length of time necessary to allowbinding of the target protein to the capture agent, thus forming thetarget protein conjugate. For example, the incubation may be at least 30minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 5hours, at least 6 hours, at least 8 hours, at least 10 hours or more.

The buffer used for the incubation can have any desired properties (pH,ionic strength, etc.) but usually mimics the buffer conditions desiredfor the eventual downstream use of the target protein. In someembodiments, the buffer conditions are those which are physiologicallytolerated in a therapeutic. In some embodiments, the pH of the buffer,and by extension the incubation, is between 4.5 and 7. The pH of thebuffer may be about 4.5, about 5, about 5.5, about 6, about 6.5 or about7. In some embodiments, the buffer has low ionic strength.

The methods may comprise measuring the absorbance of light of the targetprotein conjugate at multiple wavelengths ranging from 450 nm to about750 nm and identifying a plasmon wavelength as the wavelength at whichthere is maximal absorbance by the target protein conjugate. The plasmonwavelength (λ_(ρ)), i.e., the wavelength corresponding to maximalabsorbance shifts to greater values as the separation distance betweennanoparticles is reduced. Thus, as the target protein engages inself-association, the absorbance spectrum of the nanoparticles changesand that is reflected in the identification of the plasmon wavelength.In some embodiments, this change can be used as a binary indication ofself-association and no self-association, based on whether the plasmonwavelength changes upon absorbance measurements of the target proteinconjugates relative to the capture agent conjugates.

The sensitivity of the plasmon wavelength to changes in theinterparticle distances between antibody-gold conjugates can be used asa measure of the extent or degree of self-association of the targetprotein. In some embodiments, the methods further comprise calculatingcharge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS)score. The CS-SINS score is a ratio of the plasmon wavelength of thetarget protein conjugate with a subtraction of a plasmon wavelength froma low self-association control protein conjugate to a difference ofplasmon wavelength from a high self-association control proteinconjugate and the low self-association control protein conjugate, asdescribed in FIG. 4 (Test #2) and shown in Equation (1):

$\begin{matrix}{{{CS} - {SINS}{Score}} = \frac{\left( {{mAB} - {{parameter}1}} \right)}{\left( {{{parameter}2} - {{parameter}1}} \right)}} & (1)\end{matrix}$

wherein parameter 1 is the plasmon wavelength of a low self-associationantibody and parameter 2 is the plasmon wavelength of a highself-association antibody.

In some embodiments, a CS-SINS score less than or equal to about 0.35indicates that the target protein has favorable self-associationproperties for formulations and compositions comprising highconcentrations of the target protein (e.g., greater than or equal to 100mg/mL target protein).

In some embodiments, the method further comprises a calibration methodcomprising: capturing a plurality of calibration proteins with thecapture agent conjugate to form a plurality of calibration proteinconjugates; measuring the absorbance of light of each of plurality ofcalibration protein conjugates at multiple wavelengths ranging from 450nm to about 750 nm; and identifying the plasmon wavelength.

The calibration method may further comprise: calculating a CS-SINS scorefor each of plurality of calibration protein conjugates; and comparingthe CS-SINS scores for each of plurality of calibration proteinconjugates with previous calibration protein conjugate CS-SINS scoresfor linear fit. For example, CS-SINS for each of the plurality of thecalibration protein conjugates are fit to maximize the agreement of thelinear fit between the new data and previous CS-SINS scores byminimizing the following term: ((1−slope)²+(intercept)²). Thecalibration meets the criteria for a linear fit between the new andprevious data and, thus, proper calibration if:

-   -   the slope of the linear fit=0.9<x<1.1;    -   the intercept of linear fit=−0.1<x<0.1; and    -   R² of linear fit=>0.9.

The plurality of calibration proteins comprises proteins with a range ofself-association properties (e.g., opalescence and viscoelasticproperties). The calibration proteins may be the same type of protein asthe target protein. For example, if the target protein is antibody, thecalibration proteins are selected from different antibodies with varyingself-association properties. In some embodiments, each of the pluralitycalibration proteins are each individually selected from the groupconsisting of a monoclonal antibody, a polyclonal antibody, andfragments or derivatives thereof.

In some embodiments, the calibration proteins include the lowself-association control protein and the high self-association controlprotein, which the plasmon wavelength of conjugates thereof are used tocalculate a CS-SINS, as described elsewhere herein.

In some embodiments, the plurality of calibration proteins comprisesNIST Reference Antibody RM 8671 and human polyclonal antibody ChromPureHuman IgG, whole molecule. In some embodiments, the calibrationidentifies the plasmon wavelength of the NIST Reference Antibody RM 8671and the human polyclonal antibody ChromPure Human IgG, whole molecule. Aproper calibration would comprise a plasmon wavelength of the NISTReference Antibody RM 8671of less than 533 nm and a plasmon wavelengthof the human polyclonal antibody ChromPure Human IgG, whole molecule ofless than 534 nm.

8. SYSTEMS OR KITS FOR SCREENING A TARGET PROTEIN

In a seventh aspect, the disclosure relates to systems or kits (e.g.,reagents, computer software, instruments, etc.) for screening a targetprotein in dilute concentrations for favorable self-associationproperties (e.g., opalescence and viscoelastic properties). In someembodiments, the systems comprise one or more or each of a captureagent, a positively-charged polymer, a surface (e.g., metalnanoparticle), a solution (e.g., buffer), a target protein, at least onecalibration protein, and a spectrophotometer. The descriptions providedabove for the capture agent, positively-charged polymer, surface (e.g.,metal nanoparticle), solution (e.g., buffer), and target proteinprovided elsewhere herein are also applicable to the disclosed systems.The spectrophotometer can include any variety of instruments thatmeasure light absorption and/or transmission at a variety of lightwavelengths. Individual member components of the systems or kits may bephysically packaged together or separately.

The systems can also comprise instructions for using the components ofthe systems. The instructions are relevant materials or methodologiespertaining to the systems. The materials may include any combination ofthe following: background information, list of components and theiravailability information (purchase information, etc.), brief or detailedprotocols for using the systems, trouble-shooting, references, technicalsupport, and any other related documents. Instructions can be suppliedwith the systems or as a separate member component, either as a paperform or an electronic form which may be supplied on computer readablememory device or downloaded from an internet website, or as recordedpresentation.

It is understood that the disclosed systems and kits can be employed inconnection with the disclosed methods.

9. EXAMPLES Materials and Methods

Immunogold conjugate preparation for AC-SINS in PBS Goat-antihumanFcγ-specific antibody (Jackson ImmunoResearch Laboratories, 109-005-008)was buffer exchanged twice using Zeba desalting columns (Thermo FisherScientific, PI-89882) into 20 mM acetate (pH 4.3). The concentration wasdetermined using UV absorbance at 280 nm and a mass extinctioncoefficient of 1.26 mL/mg*cm. The antibody was diluted to 0.4 mg/mL in20 mM acetate (pH 4.3). One milliliter of 20 nm gold nanoparticles(7.0×10¹¹ particles/mL; Ted Pella Inc., 15705) was sedimented in 1.5 mLmicrocentrifuge tubes (1615-5500, USA Scientific) at 21130 rcf for 6min. Next, 950 μL of supernatant were removed and replaced with 950 μLmilliQ water. The resuspended particles were further diluted by adding500 μL of milliQ water (final concentration of 4.67×1011 particles/mL).To 100 μL of prepared capture antibody, 900 μL of gold nanoparticleswere added. The mixtures were incubated at room temperature overnight.Prior to use, the immunogold conjugates were sedimented at 21130 rcf for6 min. Nine hundred fifty microliters of supernatant were removed andreserved, and the particles were resuspended in the remainingsupernatant. The volume was carefully readjusted to 50 μL.

Preparation of polylysine-stabilized immunogold conjugates for CS-SINSGoat-antihuman Fcγ-specific antibody (Jackson ImmunoResearchLaboratories, 109-005-008) was buffer exchanged as described above anddiluted to a final concentration of 0.8 mg/mL in 20 mM acetate (pH 4.3).Polylysine (Fisher Scientific, ICN19454405), initially dissolved inmilliQ water at 5 mg/mL was diluted to either 0.8 mg/mL (0.03polylysine/IgG fraction), 2.67 mg/mL (0.10 polylysine/IgG fraction), or4.0 mg/mL (0.15 polylysine/IgG fraction) in 20 mM acetate (pH 4.3). Fora 100 μL conjugate preparation, 48.5 μL of capture IgG was mixed with1.5 μL of polylysine and reserved. Twelve hundred microliters of 20 nmgold nanoparticles (Ted Pella, 15705) were sedimented at 21130 rcf for 6min, after which 1150 μL of supernatant was removed. The nanoparticleswere resuspended in the remaining supernatant and the volume wascarefully adjusted to 50 μL. The nanoparticles were then added to theIgG/polylysine mixture and rapidly mixed by pipetting up and down 20times. The conjugates were incubated at room temperature overnight. Thedescribed preparation is for 100 μL of conjugates but can be scaled toprepare larger volumes if desired. In experiments where non-captureantibody was used, goat polyclonal IgG (Jackson ImmunoResearchLaboratories, 005-000-003) was substituted for goat-antihuman IgG.

Dynamic light scattering and zeta potential measurements of immunogoldconjugates Dynamic light scattering (DLS) experiments were performedusing the Zetasizer Nano ZSP (Malvern Panalytical, Worcestershire, UK).To assess the size of immunogold conjugates in a given buffer, 950 μL ofbuffer were added to 50 μL of the conjugates and immediately transferredto folded-capillary zeta cells (DTS1070, Malvern Panalytical). Thecuvettes were transferred to the Zetasizer instrument and the size wasmeasured at 25° C. using DLS with 173° backscatter measurements. Thesize measurements were an average of at least thirty 10 s measurements.In cases where the samples were monodisperse (immunogold conjugatesprepared for standard AC-SINS implementations), z-the average diametercalculated using cumulants analysis was reported. In cases where thesamples were polydisperse (polylysine stabilized immunogold conjugates),the major peak of the size distribution as determined by non-negativeleast squares analysis was reported. Immediately following particlesizing, the zeta potential of the particles was measured using laserdoppler velocimetry on the same instrumentation. Data was collected andanalyzed using Zetasizer 7.11 Software (Malvern Panalytical).

CS-SINS assay in histidine Antibodies of interest were buffer exchangedtwice using Zeba desalting columns (Thermo Fisher Scientific, P189882)into 10 mM histidine (pH 6.0). The concentration was determined using UVabsorbance at 280 nm and a mass extinction coefficient uniquelycalculated for each monoclonal antibody [1.40 m L/mg*cm was used forhuman polyclonal antibody (Jackson ImmunoResearch Laboratories,009-000-003)]. The antibodies were diluted to 11.1 μg/mL in histidine(pH 6.0) prior to use in CS-SINS. To a transparent and flat-bottom384-well polystyrene plate (Thermo Fisher Scientific, 12565506), 5 μL ofimmunogold conjugate were added in triplicate for each mAb assayed andfor human polyclonal antibody. Next, 45 μL of each antibody was added tothe immunogold conjugates and mixed by pipetting up and down 10 times(final antibody concentration of 10 μg/mL). The mixtures were coveredand incubated at room temperature for 4 h, after which the absorbancewas measured from 450-650 nm in 1 nm increments (normal scanning speed,8 reads per well) using a BioTek Synergy Neo plate reader (BioTek,Winooski, Vt.). The plasmon wavelength was determined by fitting asecond order polynomial to 40 data points around the observed maximumabsorbance and setting the first derivative to 0. The CS-SINS score wascalculated as described below.

Calibration of CS-SINS assay The CS-SINS measurements were calibratedand evaluated in the following manner. First, each new batch ofgold-anti Fc conjugates was evaluated using two control antibodies,namely human polyclonal antibody (ChromPure Human IgG, whole molecule,Jackson ImmunoReasearch Laboratories 009-000-003) and the NIST mAb RM8671 (NIST mAb) (See, Karageorgos, I., et al., Biologicals 2017, 50,27-34, incorporated herein by reference in its entirety). If the plasmonwavelengths for these conjugates with adsorbed human antibody weregreater than 534 nm (human polyclonal antibody) or 533 nm (NIST mAb),the experiments were terminated and the conjugates were prepared againuntil they passed this test (Test #1). Second, the gold-anti-Fcconjugates that pass Test #1 were then evaluated using a panel of sixcontrol mAbs that spanned both low and high self-association behaviors(mAbs A, C, D, E, F and K in this study), and their plasmon wavelengths(in the form of CS-SINS scores) were compared to reference data for thesame antibodies. For the reference data, the CS-SINS scores werecalculated as: (plasmon wavelength for a given mAb—plasmon wavelengthfor the lowest self-association mAb such as mAb A in thisstudy)/(plasmon wavelength for the highest self-association mAb such asmAb K in this study—plasmon wavelength for the lowest self-associationmAb such as mAb A in this study). For the measurements of the panel ofmAbs in each new experiment (i.e., the calibration measurements), theCS-SINS scores were calculated as: (plasmon wavelength for a givenmAb—parameter 1)/(parameter 2—parameter 1). The two parameters were fitto minimize the sum of ((1-slope)²+(intercept)²), where the slope andintercept terms are obtained from the linear regression between thecalibration and reference CS-SINS Scores. If any of the three values ofslope, intercept or R² values for this linear fit were not within 10% ofthe ideal values (1 for slope, 0 for intercept and 1 for R²), theexperiment was terminated and conjugates were prepared again until theypassed this test (Test #2) in addition to Test #1. If the measurementspass both tests, then the CS-SINS measurements were performed on thefull panel of antibodies and the CS-SINS scores were calculated usingthe parameters fit in Test #2 (i.e., parameters 1 and 2). Thecalibration antibodies include: A, tocilizumab; C, cetuximab; D,evolocumab; E, denosumab; F, pembrolizumab; and K, omalizumab.

Viscosity and opalescence measurements The viscosity and opalescencevalues for the experimental mAb dataset were obtained from Kingsbury etal. (Sci Adv 6, eabb0372, doi:10.1126/sciadv.abb0372 (2020),incorporated herein by reference in its entirety).

Example 1 Charge Stabilization Prevents Aggregation of Antibody-GoldConjugates

To understand the origins of the problems experienced in previousattempts to use AC-SINS (FIG. 2A, left), the aggregation ofgold-antibody (anti-human IgG) conjugates was measured in dilute buffers(10 mM acetate and/or histidine) over a range of pH values (pH 4.0-6.5;FIG. 2B, left). For pH values in the range of 5.0-6.5, plasmonwavelengths as high as ˜590 nm were observed, a significant departurefrom typical values for stable conjugates (530-535 nm) in PBS (FIG. 2E).Substantial particle aggregation was observed in the range of pH 5.0-6.5as evidenced by the large apparent diameter of the conjugates detectedby dynamic light scattering. Consistent with this result, plasmonwavelengths as high as ˜590 nm were observed, a significant departurefrom typical values for stable conjugates (530-535 nm) in PBS (FIG. 2C,left). The mechanism of particle aggregation was investigated bymeasuring the zeta potential of the antibody-gold conjugates (FIG. 2D,left) and a steady decrease was observed from a maximum value +16 mV atpH 4.0, crossing zero near pH 5.7 with a minimum of −5 mV at pH 6.5. Thetrend in zeta potential with increasing pH was also consistent with thetitration of charged amino acids intrinsic to the immobilized captureantibody. These findings suggested that low net charge of the conjugatesnear pH 5.5-6 was linked to their aggregation in the pH 5-7 range.

A large, positively-charged polymer (polylysine, ≤70 kDa) wasco-adsorbed with the capture antibody during conjugate preparation.Addition of even small amounts of polylysine (e.g., 3%, FIG. 2A, middle)was sufficient to prevent conjugate aggregation over a broad pH range of4 to 6 (FIG. 2B, middle). Increasing the polylysine to 15% (FIG. 2A,right) led to further stabilization to pH 6.5 (FIG. 2B, right). Theplasmon wavelengths of the conjugates stabilized with polylysinedisplayed similar pH-dependent trends as observed for the apparent sizesof the conjugates (FIGS. 2C, middle and 2C, right). A concomitantincrease in zeta potential of the conjugates to positive values (+2 to+6 mV) was also observed over this same pH range (FIGS. 2D, middle and2D, right). The fact that low amounts of polylysine (3%) yielded a zetapotential at pH 6 (+2.3mB) that would be associated with aggregation ofconjugates in the absence of polylysine suggested an additional, stericcomponent to the observed stabilization.

Stabilized conjugates were used to measure mAb self-interactions viaCS-SINS in histidine formulations (FIG. 8 ). Seven mAbs that display awide range of self-association behaviors based on light scatteringanalysis of mAb colloidal interactions in histidine formulations wereselected. CS-SINS analysis revealed small but detectable, mAb-dependentplasmon shifts—plasmon shifts of less than <1 nm compared to greaterthan 30 nm observed for the same antibodies using conventional AC-SINSin PBS. The capture antibody was substituted with a non-specificantibody and even smaller plasmon shifts were observed for the sevenmAbs. Conjugates prepared with only polylysine prior to addition of mAbsalso resulted in extremely small plasmon shifts that were mAbindependent. The impact of polylysine size (nominal sizes of 30-70 kDaand 15-30 kDa) on the plasmon shifts was tested and similar trends wereobserved for the conjugates prepared with different sizes of polylysine(FIG. 9 ). These results indicated that the CS-SINS assay detectsmAb-specific colloidal interactions that require antibody-mediatedimmobilization and depend weakly on polylysine size.

Robust calibration methods control for the variability of results thatare possible if precise experimental protocols are not followed. Twotests for calibrating and evaluating CS-SINS measurements were developed(FIGS. 4 and 5 ). 1) Anti-Fc gold conjugates for two calibrationantibodies human polyclonal antibody and NIST mAb should result inplasmon wavelengths of <534 nm for human polyclonal antibody and <533 nmfor the NIST mAb, as shown in FIG. 5A for a successful experiment. 2) Apanel of six calibration mAbs were used to calibrate the assay and thesemeasurements were compared to reference (historical) measurementsobtained in multiple independent assays. Two parameters were fit tomaximize the linear fit between the calibration and reference data, asexplained in the Methods section and FIG. 4 , to transform the plasmonwavelengths into calibrated CS-SINS scores. If the slope, intercept andR2 values were within 10% of their ideal values, then the assay passedTest #2 and the full panel of antibodies was evaluated. For thesuccessful experiment in FIG. 5 , the assay passed Test #2 and theevaluation data for the larger panel of antibodies (without thecalibration antibodies) showed good performance relative to referencedata. The calibration protocol was applied to CS-SINS measurements madewith either 3% or 10% polylysine for a panel of 25 commercial mAbs (FIG.6A). When analyzed as plasmon wavelength shifts, there were obviousdifferences in measurements between the two polylysine conditions.However, when the calibration protocol was applied, there was excellentagreement between the two datasets (FIG. 6B). To illustrate the value ofthis calibration process, additional experiments were performed toillustrate how experiments with flawed protocols can be readilyidentified (FIGS. 5B-5C).

Example 2 mAb Self-Interaction as Prediction of Solution Behavior

To assess the utility of charge-stabilized immunogold conjugates inmeasuring mAb self-interaction and consequently predicting antibodysolution behavior, a diverse set of 56 mAbs, including 43 commercialproducts, was employed. A range of amino acid sequence-relatedproperties (pl, charge, charge asymmetry, and hydrophobicity) werecompared for mAbs within the dataset to 500 unique antibody sequences,drawn from the broader clinical landscape using the Therapeutic AntibodyDatabase (TABS) (FIGS. 3A-D). Emphasis was placed on evaluatingsequence-derived metrics of electrostatic and hydrophobic properties,both of which have been shown previously to be drivers of mAbself-interaction. Based on this analysis, the mAb set was demonstratedto be a faithful representation of key properties of mAbs from thebroader clinical antibody landscape, including their isoelectric points,net charges (pH 6) and Fv charge asymmetry parameters (Fv-CSP, pH 6).Moreover, the panel of antibodies also displayed similar hydrophobicityproperties as evaluated in terms of the Eisenberg Hydrophobicity Index(EHI), relative to the large pane of clinical-stage antibodies. Thefindings indicated that the mAb dataset was well suited for evaluatingthe general applicability of the CS-SINS assay for evaluating antibodysolution behavior.

Greater than 30% of clinical-stage mAbs, and likely a higher percentagein earlier preclinical stages, can be expected to exhibit poor solutionbehavior such as high solution viscosity and opalescence, which manifestonly at higher concentrations (>100 mg/mL). High mAb solution viscosityis particularly problematic for subcutaneous delivery viaauto-injectors, and during ultra/diafiltration purification unitoperations. High opalescence can indicate predisposition for phaseseparation and aggregation. It was recently shown that measurement ofweak, colloidal self-interactions via the diffusion interactionparameter (k_(D)) was most effective in predicting mAbs prone to highviscosity or opalescence relative to a large set of moleculardescriptors. However, the material requirements for such measurementsrender them to be implementable only at the later, candidate selectionstages of the discovery process (FIG. 1 ) with only a few (˜10)candidates. The CS-SINS assay, on the other hand, which also measuresweak colloidal interactions, is amenable to implementation duringcandidate optimization for screening 100s to 1000s of variants (FIG. 1).

CS-SINS was conducted in ultra-dilute solutions (10 μg/ml), to determineif it could detect problematic behaviors that were manifested at fourorders of magnitude higher antibody concentrations (>100 mg/ml). Viscous(>30 cP) antibodies consistently had higher CS-SINS scores thanwell-behaved mAbs with low viscosity and that mAbs with extremeopalescence profiles (>20 NTU) were easily identified using CS-SINS(FIG. 3F). With a threshold value of 0.35, CS-SINS identifiedwell-behaved mAbs (<30 cP, <12 NTU) with >85% accuracy (FIG. 3G-3H).Setting this CS-SINS score threshold facilitated the identification ofall 10 (100%) viscous antibodies and 3 of 7 (43%) opalescent antibodies.Additionally, this threshold only les to the mischaracterization of 4 ofthe remaining 39 (10%) well-behaved antibodies. Of the 56 mAbs, 35 of 39were correctly identified as well-behaved and 13 of 17 were identifiedas having poor solution properties, comparable to classification by kip.The CS-SINS assay was also robust with respect to reproducibility,supported by detailed calibration and system suitability criteria (FIGS.3E, 4, and 5 ).

Previous work using a similar panel of antibodies revealed a strongrelationship between the diffusion interaction parameter (kip) and poorsolution properties. Further, as identified herein IgG subclass-specificbehavior with respect to the utility of kip in predicting poor solutionproperties, particularly with respect to opalescence. The relationshipbetween kip and the CS-SINS scores measured herein was explored (FIG. 7). Without segregating mAbs by subclass, a moderately strong rankcorrelation was observed between kip and CS-SINS score (Spearman's ρ of−0.80; FIG. 7A, top). However, when the mAbs were divided into IgG1/IgG2versus IgG4 subclasses, subclass-specific behavior emerged. There was amarked increase in the rank correlation for the IgG1/IgG2 grouping(Spearman's ρ of −0.89; FIG. 7A, middle) and a decrease for the IgG4subclass (Spearman's ρ of −0.76; FIG. 7A, bottom). Extending thisanalysis to the prediction of poor solution behaviors, it was observedthat using a CS-SINS threshold of 0.35 facilitated the detection of allpoorly behaved molecules in the IgG1/IgG2 subclass grouping (FIG. 7B).Applying the same standard to the IgG4 grouping revealed that all of theopalescent molecules with low CS-SINS scores belonged to this subclass(FIG. 7C), suggesting that the mode of IgG4 self-association leading toits opalescent behavior may be unique relative to IgG1/2 antibodies.More generally, these results collectively demonstrated the ability ofCS-SINS to identify antibodies with high levels of viscosity andopalescence, especially for IgG1 and IgG2 mAbs, using ultra-dilute (10μg/mL) solution measurements of self-association. The assay is robustand readily implementable for simultaneous, high-throughput screening oflarge numbers (˜100-1000) of mAb candidates. The CS-SINS assayrepresents a major advance towards identification of developableantibodies with drug-like properties in early discovery.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the disclosure, which is defined solely bythe appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and may be made without departingfrom the spirit and scope thereof.

1. A method of stabilizing a nanoparticle comprising a capture agent onthe surface, the method comprising conjugating a positively-chargedpolymer to the nanoparticle, wherein the positively-charged polymer is ahomopolymer of a positively charged amino acid or a heteropoiymer of twoor more positively charged amino acids.
 2. The method of claim 1,wherein the nanoparticle is stabilized in a solution.
 3. A nanoparticlecomprising on the surface a capture agent and a positively-chargedpolymer, wherein the positively-charged polymer is a homopolymer of apositively charged amino acid or a heteropolymer of two or morepositively charged amino acids.
 4. The nanoparticle of claim 3, whereinthe nanoparticle is comprised in a solution.
 5. A solution comprising aplurality of the nanoparticle of claim
 3. 6. A kit comprising ananoparticle and a positively-charged polymer, wherein thepositively-charged polymer is a homopolymer of a positively chargedamino acid or a heteropolyrner of two or more positively charged aminoacids.
 7. The kit of claim 6, further comprising a solution and/or acapture agent.
 8. A method for determining the tendency of a targetprotein to self-associate, comprising: (i) capturing the target proteinin the solution of claim 5; and (ii) determining the color of thesolution, wherein a change in the color of the solution compared to acontrol solution without the target protein indicates a tendency of thetarget protein to self-associate.
 9. A method for screening a targetprotein in dilute concentrations for self-association properties,comprising: adsorbing the capture agent and the positively-chargedpolymer onto the nanoparticle in the kit of claim 7 to form a captureagent conjugate; incubating the capture agent conjugate with the targetprotein in a solution to form a target protein conjugate; measuring theabsorbance of light of the target protein conjugate at multiplewavelengths ranging from 450 nm to about 750 nm; and identifying aplasmon wavelength as the wavelength at which there is maximalabsorbance by the target protein conjugate, wherein thepositively-charged polymer is a homopolymer of a positively chargedamino acid or a heteropolyiner of two or more positively charged aminoacids.
 10. A system for screening a target protein in diluteconcentrations for self-association properties according to the methodof claim 9, said system comprising one or more or each of: a captureagent; a positively-charged polymer, wherein the positively-chargedpolymer is a homopolymer of a positively charged amino acid or aheteropolymer of two or more positively charged amino acids; ananoparticle; a solution; a target protein; at least one calibrationprotein; and a spectrophotometer.
 11. The method of claim 1, wherein thenanoparticle is a metal nanoparticle.
 12. The method of claim 1, whereinthe positively-charged polymer is polylysine.
 13. The method of claim 1,wherein the capture agent is an antibody.
 14. The method of claim 8,wherein the target protein is an antibody.
 15. The method of claim 2,wherein the solution has a low ionic strength and/or a pH of 3.5-7. 16.The method of claim 11, wherein the metal nanoparticle is a goldparticle.